Ab32027

Total protein was extracted with RIPA buffer (Solarbio). After the protein was quantified using BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA), the equal amounts of protein samples were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% fat-free milk for 2 h and incubated with primary antibodies against E-cadherin (1:500, ab15148, Abcam), Vimentin (1:2,000, ab137321, Abcam), CBL (1:5,000, ab32027, Abcam), or β-actin (1:2,000, ab8227, Abcam). After washing thrice with tris-buffered saline, the membranes were probed with horseradish peroxidase-labeled secondary antibody (1:25,000, ab205718, Abcam). Finally, the protein bands were measured using the ECL system (Beyotime, Shanghai, China).

Total cell and tissue lysates were prepared in 1× sodium dodecyl sulphate (SDS) buffer. Identical quantities of protein were separated by SDS gel electrophoresis and transferred onto nitrocellulose filter membranes. After incubating with antibodies specific for QPCT (ab201172, Abcam, CA, USA) and GAPDH (sc-25778; Santa Cruz, CA, USA), the blots were incubated with IRDye 800-conjugated goat anti-rabbit IgG, and bands were detected using an Odyssey infrared scanner (Li-Cor). GAPDH was used as the loading control. The other antibodies used for western blot were against IRS1 (ab52167, Abcam, CA, USA), NF-κB (p65) (8242, Cell Signalling Technology), HRAS (ab32417, Abcam, CA, USA), CBL (ab32027, Abcam, CA, USA), GAB1 (ab59362, Abcam, CA, USA), NAF1 (ab157106, Abcam, CA, USA), MAPK8 (ab199380, Abcam, CA, USA), MAPK10 (ab126591, Abcam, CA, USA), FAK (PTK2) (ab40794, Abcam, CA, USA), p-FAK (ab81298, Abcam, CA, USA), ERK1/2 (4695, Cell Signaling Technology), p-ERK1/2 (4370, Cell Signaling Technology), AKT (4691, Cell Signaling Technology), p-AKT (4060, Cell Signaling Technology), Stat3 (9139, Cell Signaling Technology), p-Stat3 (9145, Cell Signaling Technology), and ubiquitin (3936, Cell Signaling Technology).